Method for the cultivation of primary cells and for the amplification of viruses under serum free conditions

ABSTRACT

The present invention relates to a method for the cultivation of primary cells. The primary cells are cultivated in a serum free medium comprising a factor selected from the group consisting of growth factors and attachment factors. 
     The method for the cultivation of primary cells may be one step in a method for the amplification of viruses, such as poxviruses. According to this latter method the primary cells are cultivated in a serum free medium comprising a factor selected from the group consisting of growth factors and attachment factors. The cells are then infected with the virus and the infected cells are cultivated in serum free medium until progeny virus is produced.

FIELD OF THE INVENTION

The present invention provides a method for the cultivation of primarycells. The primary cells are cultivated in a serum free mediumcomprising a factor selected from the group consisting of growth factorsand attachment factors. The method for the cultivation of primary cellsmay be one step in a method for the amplification of viruses, whereinthe cells are then infected with a subject virus and the infected cellsare cultivated in serum free medium until progeny virus is produced. Theinvention encompasses the cultivation of poxviruses according to thismethod.

BACKGROUND OF THE INVENTION

Most viral vaccines such as attenuated or recombinant viruses aremanufactured from cell culture systems. The cells used for virus/vaccineproduction may be cell lines, i.e. cells that grow continuously invitro, either as single-cell suspension culture in bioreactors or as amonolayer on a cell-support surface of tissue culture flasks orroller-bottles. Some examples for cell lines used for the production ofviruses are: the human fetal lung cell-line MRC-5 used for themanufacture of polio viruses and the human fetal lung cell-line WI-38used for the manufacture of measles virus, mumps virus and rubella virus(MMR II) (Merck Sharp & Dohme).

Not only cell lines but also primary animal cells are used for themanufacture of vaccines. An example of primary cells that are used forvirus production are chicken embryo fibroblasts (CEF cells). These cellsare used for the production of measles and Japanese encephalitis virus(Pasteur Merieux), mumps virus (manufactured by Provaccine), rabiesvirus (manufactured by Chiron Berhing GmbH & Co.), yellow fever virus(manufacture by Aprilvax), influenza virus (manufactured by Wyeth Labsand SmithKline & Beecham) and modified Vaccinia virus Ankara (MVA).

CEF cells are often used since many virus vaccines are made byattenuating the virulent disease-causing virus by serially passaging inCEF cells. The attenuated virus does not longer cause the disease but isstill capable of stimulating a potent protective immunity against thevirulent form of the virus. An example for this type of virus is MVA.This virus is severely replication restricted in humans and in mostanimals. MVA is being developed as a vaccine vector because it can beused to express antigens derived from a variety of agents causingdiseases in humans. Attenuated viruses, such as MVA are preferably notpropagated on human cells since there is a concern that the virusesmight become replication competent in cells of human origin. Virusesthat have regained the ability to replicate in human cells represent ahealth risk if administered to humans, in particular if the individualsare immune compromised. For this reason, some attenuated viruses, suchas MVA, are strictly manufactured from CEF cells, if intended for humanuse.

Moreover, CEF cells are used for those viruses that grow only on saidcells. Examples of such viruses are avian viruses such as avipoxviruses, canary pox virus, ALVAC, Fowl pox virus and NYVAC.

Cell lines and primary cells grown under in vitro culturing conditionsrequire a special growth and maintenance medium that can support (I)cell replication in the logarithmic phase and (II) cell maintenance oncethe cells are no longer dividing, i.e., when the cells are in thestationary phase. The commonly used cell culture media comprise a richsalt solution containing vitamins, amino acids, essential trace elementsand sugars. Growth hormones, enzymes and biologically active proteinsrequired for supporting cell growth and maintenance are usually added asa supplement to the medium in the form of an animal blood derived serumproduct. Examples of animal blood derived serum products are fetal calfserum, chicken serum, horse serum and porcine serum. These sera arederived from fractionated blood, from which the red blood cells and thewhite blood cells have been removed. Primary cells, such as CEF cellsare even more dependant on animal serum sources than cell lines. Thus,primary cells are usually cultivated in cell culture media comprising 5to 10% serum, in most cases fetal calf serum (FCS).

The animal sera not only comprise factors that are required for thegrowth of cells, but also factors that are required for cells thatnaturally grow as adherent cells to attach to the cell support surfaceof the culture vessel. Thus, it is critical for adherent cells thatenough serum is added to the medium to enable them to grow and form amonolayer.

Unfortunately, bovine/fetal calf serum as well as sera from otheranimals may contain adventitious pathogenic agents such as viruses orprion proteins. There is a potential risk that these pathogenic agentsmay be transmitted to the animal/human to be treated or vaccinated withthe vaccine or any other pharmaceutical product produced in cellculture. This is of particular relevance if cell culture products areadministered to immune-compromised humans. One of the many potentialmajor problems associated with the commonly used bovine serum supplementis the possibility to transmit the agent causing bovine spongiformeencephalopathy (BSE) to the animals/humans that come into contact withthe products produced from cell culture.

In view of the possible risk associated with the use of animal sera incell culture it has become clear that manufacturing processes free fromthe use of animal products are highly desirable.

To this end, specific media that do not have to be supplemented withanimal sera have been developed for continuously growing cell lines andfor the production of viruses in continuously growing cell lines,respectively. An example of such a serum free medium that can be used tocultivate cell lines is VP-SFM manufactured by Gibco BRL/LifeTechnologies. According to the manufacturer's information VP-SFM isdesigned specifically for the growth of VERO, COS-7, MDCK, Hep2, BHK-21and other important cell lines (Price, P. and Evege, E. Focus 1997, 19:67-69) and for virus production in said cell lines. No information isavailable regarding the cultivation of primary cells in the medium.

THE PRESENT INVENTION Summary of the Invention

What we therefore believe to be comprised by our invention may besummarized inter alia in the following words:

A method for the amplification of a virus comprising:

-   -   cultivating primary avian cells permissive for productive        replication of the virus in a serum free medium comprising a        factor selected from the group consisting of growth factors and        attachment factors,    -   infecting of the primary avian cells with the virus,    -   cultivating the infected cells in serum free medium until        progeny virus is produced, and    -   isolating the virus from the culture; such a

method, wherein the serum free medium comprising growth factors andattachment factors is removed at the time of infecting the primary aviancells with the virus, and/or during cultivating of the infected cellsuntil virus progeny is produced, and replaced with a serum free mediumwhich does not comprise growth factors and attachment factors; such a

method wherein, subsequent to cultivating the infected cells in serumfree medium until progeny virus is produced, one or more viruspurification steps are performed; such a

method wherein the virus used for infection of primary avian cells waspreviously propagated or may have been previously propagated in thepresence of animal sera and is subsequently re-derived through severalrounds of plaque purification by limited dilution in serum free mediumto reduce the risk of serum contamination; such a

method which is repeated at least once to obtain a virus or virus stockwhich is essentially free of products and/or infectious agents comprisedin animal sera; such a

method wherein the primary avian cells are Chicken Embryo Fibroblasts(CEF); such a

method wherein the growth factor is an epidermal growth factor (EGF);such a

method wherein the epidermal growth factor (EGF), is recombinant-humanEGF; such a

method wherein the concentration of EGF is in a range of 5 to 20 ng/mlmedium; such a

method wherein the attachment factor is fibronectin; such a

method wherein the concentration of fibronectin is in the range of 1 to10 ug/cm² surface of the cell culture vessel; such a

method wherein the medium comprises two or more factors selected fromgrowth factors and attachment factors; such a

method wherein the medium comprises EGF and fibronectin in concentrationranges of 5 to 20 ng/ml and 1 to 10 ug/ml medium, respectively; such a

method wherein the medium further comprises one or more additivesselected from a microbial extract, a plant extract and an extract from anon-mammalian animal; such a

method wherein the microbial extract is a yeast extract or a yeastolateultrafiltrate; such a

method wherein the plant extract is a rice extract or a soya extract;such a

method wherein the extract from a non-mammalian animal is a fishextract; such a

method wherein the virus is selected from mumps virus, measles virus,rabies virus, Japanese encephalitis virus, yellow fever virus, influenzavirus and poxvirus; such a

method wherein the poxvirus is an attenuated virus or a recombinantvirus; such a

method wherein the poxvirus is an orthopoxvirus; such a

method wherein the orthopoxvirus is a Vaccinia virus; such a

method wherein the Vaccinia virus is Modified Vaccinia virus Ankara;such a

method wherein the Modified Vaccinia virus Ankara is selected fromMVA-575 (ECACC V00120707), MVA-572 (ECACC V94012707), and MVA-BN (ECACCV00083008), or a derivative of such virus; such a

poxvirus obtained by:

-   -   cultivating primary avian cells permissive for productive        replication of the virus in a serum free medium comprising a        factor selected from the group consisting of growth factors and        attachment factors,    -   infecting the primary avian cells with the virus,    -   cultivating the infected cells in serum free medium until        progeny virus is produced, and    -   isolating the virus from the culture; such a

poxvirus wherein the primary avian cells are Chicken Embryo Fibroblasts(CEF); such a

poxvirus wherein the virus used for infection of primary avian cells waspreviously propagated or may have been previously propagated in thepresence of animal sera and which virus is subsequently re-derivedthrough several rounds of plaque purification by limited dilution inserum free medium; such a

poxvirus wherein 4-6 rounds of plaque purification are performed; such a

poxvirus wherein the risk of the poxvirus to contain a BSE particle isless than 10³²; such a

poxvirus which is essentially free of any products and/or infectiousagents comprised in animal sera; such a

poxvirus wherein the poxvirus is Modified Vaccinia virus Ankara; such a

poxvirus wherein the Modified Vaccinia virus Ankara is selected fromMVA-575 (ECACC V00120707), MVA-572 (ECACC V94012707), and MVA-BN (ECACCV00083008), or a derivative of such virus; such a

poxvirus wherein the poxvirus is an attenuated virus or a recombinantvirus; such a

vaccine comprising the poxvirus; such a

pharmaceutical composition comprising the poxvirus and apharmaceutically acceptable carrier, diluent and/or additive; such a

pharmaceutical composition which is essentially free of any productsand/or infectious agents comprised in animal sera; such a

method for the treatment of an animal, including a human, in needthereof, comprising administering to the animal, including a human, thepoxvirus; such a

method wherein the poxvirus is administered as a vaccine; such a

method for the treatment of an animal, including a human, in needthereof, comprising administering to the animal, including a human, thepharmaceutical composition; such a

method for the vaccination of an animal, including a human, comprisingadministering to the animal, including a human, the vaccine; such a

method for enhancing a specific immune response to a vaccine in a livingmammal, including a human, comprising administering a vaccine and anadjuvant-effective amount of the poxvirus.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method for cultivation of primarycells, in particular primary avian cells, in serum free medium and amethod for the production of virus in primary cells under serum freeconditions. The instant method for the cultivation of primary cells maybe characterized in that the cells are cultivated in a serum free mediumcomprising a factor selected from the group consisting of growth factorsand attachment factors.

According to the present invention primary cells that naturally grow asadherent cells attach to the surface of the cell culture vessel afterseeding and grow in a logarithmic phase until a monolayer is formed.According to the present invention the resting cells may be maintainedin the medium used during the attachment and logarithmic growth of thecells.

The term “primary cells” as used in the present description is wellknown to a person skilled in the art. Without being restricted to thefollowing definition the term “primary cells” may refer to cells thathave been freshly isolated from an animal or human tissue, organ ororganism, wherein the cells are not able to continuously andindefinitely replicate and divide. Usually, primary cells divide in cellculture less than 100 times, often less than 50 times, often less than25 times. Thus, primary cells have not undergone an immortalizing event.Examples for primary cells are cord blood lymphocytes and human oranimal fibroblasts. Representative examples of animal fibroblasts areavian fibroblasts, such as Chicken Embryo Fibroblasts (CEF cells). Anexample of primary human fibroblasts is human foreskin fibroblasts.

Methods of isolating primary cells are known. Generally, primary cellcultures are derived directly from tissues, organs or embryos. Thetissues, organs or embryos are subjected to protease treatment to obtainsingle cells. The cells are then cultivated according to the method ofthe present invention under in vitro culture conditions.

More specifically, CEF cells are obtained from protease digested chickenembryos. CEF cells grow best as adherent cells attached to a solid cellsupport surface. The cells start replication and establish a monolayer.If CEF cells (after embryo digestion) are cultivated in vitro with astandard culturing medium and without animal serum, the cells willoccasionally attach to the solid cell-support surface, but will notreplicate to form a confluent monolayer of cells and will, with time,slowly detach from the solid culturing-support surface. In contrast, ifthe CEF cells are cultivated according to the method of the presentinvention, the cells attach to the solid support, grow in thelogarithmic phase until a monolayer is formed and can be maintained inthe stationary phase for several days.

The method of the present invention is not restricted to cells that formmonolayers. According to an alternative embodiment the method accordingto the present invention may be used for all other types of primarycells, such as cells naturally growing in suspension culture (e.g.lymphocytes or other types of blood cells) or cells that naturally wouldgrow as adherent cells but have been adapted to growing in suspensionculture.

As shown below the cells can also be used for the serum freeamplification of viruses that might be useful as vaccines.

Viruses, including e.g. wild-type viruses, attenuated viruses andrecombinant viruses that are used as vaccines, may be amplified underserum containing conditions. However as noted above, there is apotential risk that serum contains pathogenic agents (such as TSE/BSE)may be transmitted to the animal/human treated or vaccinated with thevaccine. To reduce the risk of contaminants in the vaccine, it is afurther aspect of the invention to passage and/or cultivate and/orplaque purify and/or purify by limited dilution or any other methodunder serum free conditions those viruses that previously have beenamplified or may have been previously amplified under serum containingconditions and that have been used or are intended to be used asvaccine. A virus that may by used in a vaccine and that is passagedand/or cultivated and/or plaque purified and/or purified by limiteddilution or any other method under serum free conditions may be awild-type virus, an attenuated virus or a recombinant virus.

It was unexpected that primary cells naturally growing as adherent cells(I) can effectively attach to the surface of the cell culture vesselwithout forming unacceptable amounts of aggregates and (II) can be grownin the logarithmic phase in the absence of serum since it is generallybelieved that primary cells are dependant on a multitude of differentfactors and components comprised in serum. Moreover, it is believed thatadherent cells form non-viable aggregates that do not attach to thesurface of the cell culture vessel, when cultivated in serum freemedium. Thus, it was unexpected that it is sufficient to add to a serumfree medium a factor selected from the group consisting of growthfactors and attachment factors in order to obtain attachment and growthof adherent primary cells. Moreover, it was also unexpected that primarycells cultivated in suspension culture can be grown with the media usedin the method according to the present invention.

Furthermore, it was surprising that primary avian cells, such as theinstant Chicken Embryo Fibroblasts (CEF), can be cultivated to attach tothe surface of a cell culture vessel without forming unacceptableamounts of aggregates in a serum free medium comprising a factorselected from the group consisting of growth factors and attachmentfactors. Avian cells are otherwise understood to grow adversely in serumfree medium not comprising growth factors or attachment factors, i.e.,it was unexpected that the poor growth properties of primary avian cellscould be improved significantly by adding a factor selected from growthfactors and attachment factors to serum free medium.

The term “cultivation of cells” in a serum free medium in the context ofadherent primary cells refers to the seeding of the cells into theculture vessel in a serum free medium, to the growing of the cells in aserum free medium in the logarithmic phase until a monolayer is formedand/or to the maintenance of the cells in serum free medium as soon asthe monolayer is formed. The term “cultivation of cells” in a serum freemedium also refers to a method in which all of the above mentioned stepsare performed with serum free medium, so that no animal serum productsare present during the whole cultivation process of the cells. Thus, ina more general meaning the term “cultivation of cells in a serum freemedium” refers to the fact that all media leading to the formation of amonolayer are serum free media. The media used in all of the above stepsmay comprise a factor selected from growth factors and attachmentfactors. However, it might be sufficient to add such a factor only tothe media used for the attachment of the cells and/or the growing of thecells under logarithmic conditions.

The term “cultivation of cells” in a serum free medium in the context ofcells growing in suspension culture refers to the seeding of the cellsinto the culture vessel in a serum free medium, the growing of the cellsin a serum free medium in the logarithmic phase and/or the maintenanceof the cells in serum free medium as soon as the saturation density atwhich no further replication occurs is obtained. The term “cultivationof cells” in a serum free medium refers to a method in which all of theabove mentioned steps are performed with serum free medium, so that noanimal serum products are present during the whole cultivation of thecells. The media used in all of the above steps may preferably comprisea factor selected from the group of growth factors. However, it might besufficient to add such a factor only to the media used for the seedingof the cells and/or the growing of the cells under logarithmicconditions. As explained below in more detail it might also be possibleto cultivate cells that would normally grow as attached cells also assuspension culture cells if appropriate incubation conditions are chosen(e.g. by applying “wave” incubation). The method according to thepresent invention also applies for this type of incubation.

The term “serum-free” medium refers to any cell culture medium that doesnot contain sera from animal or human origin. Suitable cell culturemedia are known to the person skilled in the art. These media comprisesalts, vitamins, buffers, energy sources, amino acids and othersubstances. An example of a medium suitable for the serum freecultivation of CEF cells is medium 199 (Morgan, Morton and Parker; Proc.Soc. Exp. Biol. Med. 1950, 73, 1; obtainable inter alia fromLifeTechnologies).

The media used according to the method of the present invention, inparticular the media used for adherent cells such as CEF cells, containa factor selected from the group consisting of growth factors andattachment factors. An example of an attachment factor is fibronectin.

For cells that naturally grow as adherent cells, which, however, arenevertheless cultivated in suspension culture (which is possible e.g.for CEF cells), it is a further aspect of the invention to use a factorselected from growth factors. Examples of growth factors useful for thistype of cultivation are recombinant bovine, mouse, chicken, humanepidermal growth factor (EGF), particularly recombinant human EGF(rh-EGF) (Chemicon Int., catalog number: GF001).

For cells naturally growing in suspension culture the medium maycomprise a factor selected from the group of growth factors includingEGF. Growth factors for these types of cells are factors specific fornon-adherent cells. Examples of these growth factors are interleukins,GM-CSF, G-CSF and others. The person skilled in the art may easilydetermine by routine experimentation, which type of factor is suitablefor which type of cells.

If the factor added to the serum free medium is EGF, in particularrh-EGF, it is an aspect of the invention to add such growth factor tothe medium at a concentration of 1 to 50 ng/ml. It is a further aspectto add such factor at a concentration of 5 to 20 ng/ml. However, theperson skilled in the art will be aware of the fact that different celltypes may require a somewhat different concentration of EGF in themedium for optimal results.

If the attachment factor added to the serum free medium is fibronectin:(e.g. Chemicon Int.; Human plasma fibronectin; catalog number FC010), itis an aspect of the invention to add such factor to the medium at aconcentration of 1 to 50. It is a further aspect to add such factor at aconcentration of 1 to 10 μg/cm² surface of the cell culture vessel.However, those skilled in the art understand that different cell typesmay require a somewhat different concentration of fibronectin in themedium for optimal results.

It is sufficient to add only one factor selected from growth factors andattachment factors to the medium, in particular if the cells areadherent cells. However, it is also possible to add two or more factorsselected from growth factors and attachment factors to the medium. Themedium may comprise EGF and fibronectin, possibly in the concentrationranges defined above, in particular if the primary cells are adherentcells such as CEF cells.

The medium may further comprise one or more additives selected frommicrobial extracts, plant extracts and extracts from non-mammaliananimals. The microbial extract may be a yeast extract or yeastolateultrafiltrate. The plant extract may be a rice extract or soya extract.The extract from non-mammalian animals may be a fish extract.

Asparagine may also be added to the commercially available serum freemedium to which a factor selected from growth factors and attachmentfactors has been added. Asparagine may also be added to the medium thatis used during the infection with virus (see below). Commercial serumfree media usually comprise asparagine in a concentration range of 0.3to 1.0 mM. It is an aspect of the invention to add asparagine tosupplement the medium in the range of 0.5 to 1.5 mM. A 1 mM asparaginesupplement may be adequate. The total concentration of asparagine in themedium is less than 2 mM, in the range of 0.8 to 1.8 mM. For example,the concentration of asparagine in the medium is 1.3 mM.

Moreover, glutamine may also be added to the medium. Glutamine may alsobe added to the medium that is used during the infection with virus (seebelow). Glutamine may also be added to supplement the medium atconcentrations in the range of 1 to 5 mM. It is a further aspect of theinvention to add glutamine at a concentration in the range of 2 to 4 mM.The indicated ranges also correspond to the total concentrations in themedium since most of the commercially available media do not containglutamine.

Amplification of a virus may comprise the following steps: in the firststep primary cells are cultivated according to the method describedabove, i.e. primary cells are cultivated in a serum free mediumcomprising a factor selected from the group consisting of growth factorsand attachment factors, depending on the cell type. All conditions anddefinitions given in the description of the method for the cultivationof primary cells above also apply to the definition of the first step ofthe method for the amplification of virus according to this embodimentof the present invention. In a second step the primary cells areinfected with the virus. In the third step the infected cells areincubated in serum free medium until progeny virus is produced. Finally,in a fourth step, the virus is isolated from infected cells.

The term “amplification of a virus” is used to make clear that themethod according to the present invention is typically used to increasethe amount of virus due to a productive viral replication of the virusin the infected cells. In other words the ratio of output virus to inputvirus should be above 1. Primary cells are chosen for a specific virusin which the virus is able to productively replicate. The term“reproductive replication” refers to the fact that the specific virusreplicates in the specific primary cell to such an extent thatinfectious progeny virus is produced, wherein the ratio of output virusto input virus is above 1.

The selection of primary cell type which supports productive replicationof a particular virus is known. By way of example the primary cells maybe human foreskin fibroblasts if the virus to be amplified is the humanCytomegalovirus; the primary cells may be CEF cells if the virus to beamplified is measles virus, mumps virus, rabies virus, Japaneseencephalitis virus, yellow fever virus, influenza virus or a poxvirussuch as vaccinia virus.

Methods for infecting primary cells according to the second step ofinstant method for virus amplification are known. By way of example thevirus may simply be added to the medium. Alternatively, the medium maybe removed and the virus may be added to fresh medium, which in turn isadded to the cells. To obtain an efficient infection the amount of thevirus/medium suspension should be as low as possible to have a highvirus concentration. After the attachment of the virus additional mediummay be added.

In the third step of the instant method, the infected cells arecultivated in serum free medium until progeny virus is produced.

The serum free medium that is used in the second and third step of themethod for the amplification of a virus may be the same medium that hasalready been used before, i.e. a serum free medium comprising a factorselected from growth factors and attachment factors, depending on thecell type. Alternatively, the serum free medium comprising growthfactors and attachment factors may be removed at the step of infectingthe primary avian cells with the virus, and/or at the step ofcultivating the infected cells until virus progeny is produced, andreplaced with a serum free medium which is essentially free of growthfactors and attachment factors without adverse effects on the culture.

During all stages the medium may be supplemented with asparagine and/orglutamine as outlined above, wherein the total concentration ofasparagine in the medium is as defined above.

The progeny virus may be concentrated and purified according to methodsknown to the person skilled in the art.

Thus, the present invention relates to a method for the amplification ofa poxvirus comprising the following steps: (I) cultivating primary cellsaccording to a method as described above, i.e. a method in which theprimary cells are cultivated in serum free medium comprising a factorselected from the group consisting of growth factors and attachmentfactors, depending on the cell type, (II) infecting the primary cellswith the poxvirus, (III) cultivating the infected cells in serum freemedium until progeny virus is produced and (iv) isolating the virus fromthe infected cells. Viruses isolated according to the instant method arefree of any products and/or infectious agents comprised in animal sera.

The process of passaging and/or further cultivating and/or plaquepurifying and/or purifying by limited dilution or any other method underserum free conditions those viruses that previously have been amplifiedunder serum containing conditions is termed “re-derivation”. Such are-derivation may also be used if it is not completely clear how amaster seed was obtained that is used for the production of a vaccine. Are-derivation under serum-free drastically reduces the risk ofcontaminations in the vaccine, in particular of undesired infectiousagents.

The methods according to the present invention relate to cultivatingprimary avian cells and to amplifying a virus, wherein the virus is thevirus that is to be re-derived. For example, the instant methods areuseful for the re-derivation of viruses and virus stocks, in particularfor stocks that have been, or may have been passaged in serum containingmedium and/or with unclear passage histories.

One passage of the starting material, i.e. of the virus to be re-derivedunder serum free conditions, may be sufficient for the re-derivation ofa virus. The term “passaging” refers to the steps of cultivating cellsunder serum free conditions, infection of the cells with the virus to bere-derived and obtaining the virus produced in the infected cells.Although one passage might be sufficient, it may be preferable topassage the virus several times under serum free conditions. In thiscase the virus obtained from the first passaging step is used to againinfect fresh cells. The passaging under serum free conditions may becombined with one or more plaque purifications and/or with limiteddilution and/or any other method for the purification of a virus underserum free conditions. The total number of passages, optionally byincluding plaque purification and/or limited dilution is in a range of 1to more than 10, such as 3 to 8 or 4 to 6. The techniques of plaquepurification and/or limited dilution are standard virological methodspracticed by those skilled in the art.

The re-derivation according to the present invention may be done with avirus starting material that exhibits desired biological properties,wherein the virus used as starting material may have been amplifiedunder serum containing conditions. After several passages under serumfree conditions according to the present invention it is confirmedwhether the virus passaged under serum free conditions isidentical/similar to the virus originally passaged under serumcontaining methods. In most cases the virus obtained after there-derivation has similar/identical properties to the virus used asstarting material. There may also be situations in which the re-derivedvirus has even improved properties compared to the virus used asstarting material, e.g. an improved safety profile.

The present invention also relates to the re-derived virus obtained bythe method according to the present invention. The risk that poxvirusobtained by the re-derivation method according to the present inventioncomprises a BSE particle is less than 10³².

If it is intended to re-derive a virus, the following re-derivation planmay be used by way of example. This plan is particularly suitable forthe re-derivation of a Vaccinia virus, e.g. an MVA strain that has ormay have been cultivated under serum containing conditions: First theoriginal Master Virus Bank (MVB) virus seed stock is re-cloned through,for example, 5 rounds of plaque purification by limited dilution (seeexample 9). Viruses from the original virus seed stock and from the newre-derived stock are compared both genetically and phenotypically. Agenetic comparison of the virus cultivated under serum containingconditions and the re-derived virus may be made by, for example, (i)Restriction Enzyme mapping of the viral genome (RE-Map), (ii) PCRamplification of relevant parts of the genome such as the six deletionsites in case on MVA and/or (iii) PCR based restriction fragment lengthpolymorphism mapping of the viral genome (PCR-RFLP Assay). A phenotypiccharacterization may, for example, be performed by: (i) comparinghumoral responses in vaccinated mice, (ii) comparing efficacy using alethal vaccinia model, (iii) evaluating safety by the vaccination ofseverely immune compromised mice, (iv) comparing the attenuation(replication) in a variety of mammalian cell lines.

The present invention consequently relates to a re-derivation processnamely the method for the cultivation of primary avian cells as definedabove and/or the method for the amplification of a virus as definedabove, wherein the virus is the virus that is to be re-derived. Theinvention further relates to re-derived virus such as re-derivedVaccinia viruses, e.g. MVA strains such as MVA-BN. The re-derived viruscan be e.g. a re-derived wild type virus, a re-derived attenuated virusor a re-derived recombinant virus. The invention further relates tocompositions comprising re-derived virus.

The invention further relates to a virus including a re-derived virus,in particular to the viruses including the re-derived viruses as definedabove as a medicament or vaccine. If the virus is a wild-type virus oran attenuated virus the virus can be used for vaccination against thevirus as such. For this purpose attenuated viruses are particularlypreferred. If the virus is a recombinant virus expressing proteinsexpressed from genes heterologous to the viral genome, it is possible tovaccinate against the virus as such and/or against the expressedheterologous protein. If the recombinant virus expresses a therapeuticgene such as an antisense RNA or a ribozyme the virus may be used as amedicament.

The term “recombinant virus” refers to any virus having inserted intothe viral genome a heterologous gene that is not naturally part of theviral genome. A heterologous gene can be a therapeutic gene, a genecoding for a peptide comprising at least one epitope to induce an immuneresponse, an antisense expression cassette or a ribozyme gene. Methodsto construct recombinant viruses are known to a person skilled in theart. Poxvirus vectors such as MVA, in particular MVA 575, MVA 572, andMVA-BN may be used (see above).

An “attenuated virus” is a virus originating from a pathogenic virus butthat upon infection of the host organism leads to a lower mortalityand/or morbidity compared to the non-attenuated parent virus. Examplesof attenuated poxviruses are known to the person skilled in the art. Anexample for an attenuated Vaccinia virus is strain MVA, in particularthe strain that has been deposited at ECACC with the deposition numberV00083008 (see above).

As discussed previously, it is understood by those skilled in the artthat primary avian cells grow adversely under serum free conditions. Theadditional stress associated with a poxvirus infection may be expectedto cause the already stressed cells to die before a significantamplification of the poxvirus occurs. Surprisingly, avian cells grownaccording to the present method, in a serum free medium comprising afactor selected from growth factors and attachment factors, effectivelysupport viral replication and amplification of poxviruses.

The poxvirus is preferably an orthopoxvirus. Examples of orthopoxviruses are avipoxviruses and vaccinia viruses.

The term “avipoxvirus” refers to any avipoxvirus, such as Fowlpoxvirus,Canarypoxvirus, Uncopoxvirus, Mynahpoxvirus, Pigeonpoxvirus,Psittacinepoxvirus, Quailpoxvirus, Peacockpoxvirus, Penguinpoxvirus,Sparrowpoxvirus, Starlingpoxvirus and Turkeypoxvirus. Preferredavipoxviruses are Canarypoxvirus and Fowlpoxvirus.

An example of a canarypox virus is strain Rentschler. A plaque purifiedCanarypox strain termed ALVAC (U.S. Pat. No. 5,766,598) was depositedunder the terms of the Budapest treaty with the American Type CultureCollection (ATCC), accession number VR-2547. Another Canarypox strain isthe commercial canarypox vaccine strain designated LF2 CEP 524 24 10 75,available from Institute Merieux, Inc.

Examples of a Fowlpox virus are strains FP-1, FP-5 and TROVAC (U.S. Pat.No. 5,766,598). FP-1 is a Duvette strain modified to be used as avaccine in one-day old chickens. The strain is a commercial fowlpoxvirus vaccine strain designated O DCEP 25/CEP67/2309 October 1980 and isavailable from Institute Merieux, Inc. FP-5 is a commercial fowlpoxvirus vaccine strain of chicken embryo origin available from AmericanScientific Laboratories (Division of Schering Corp.) Madison, Wis.,United States Veterinary License No. 165, serial No. 30321.

Examples of vaccinia virus strains are the strains Temple of Heaven,Copenhagen, Paris, Budapest, Dairen, Gam, MRIVP, Per, Tashkent, TBK,Tom, Bern, Patwadangar, BIEM, B-15, Lister, EM-63, New York City Boardof Health, Elstree, Ikeda and WR. The invention is preferably carriedout with modified vaccinia virus Ankara (MVA) (Sutter, G. et al. [1994],Vaccine 12: 1032-40). Typical MVA strains are MVA 575 that has beendeposited at the European Collection of Animal Cell Cultures under thedeposition number ECACC V00120707 and MVA-572 deposited at ECACC underthe deposition number V94012707. MVA-BN has been deposited at theEuropean Collection of Animal Cell Cultures with the deposition numberECACC V00083008.

The virus to be amplified according to the method of the presentinvention may be a wild-type virus, an attenuated virus or a recombinantvirus.

As pointed out above, for poxviruses the primary cells may be primaryavian cells such as CEF cells or primary duck embryo fibroblasts. Again,one skilled in the art understands which primary cells are suitable forthe amplification of which poxvirus. CEF cells are known for theamplification of MVA. If the method according to the present inventionis used for the amplification of MVA in CEF cells, the starting pH ofthe medium may be in a range of about 7.0 to about 8.5. For MVAamplification in CEF cells in serum free medium, it is an aspect of theinvention to select one or two of the factors selected from EGF andfibronectin.

The invention further refers to viruses, in particular poxvirusesobtained by the above-described method. According to a preferredembodiment the poxvirus is a vaccinia virus, most preferably a MVAstrain such as MVA-BN.

The invention further concerns a composition comprising a virus, inparticular a poxvirus produced by the method according to the presentinvention. As pointed out above the poxvirus is preferably a vacciniavirus, most preferably a MVA strain such as MVA-BN. Due to the methodfor amplification of the virus the composition is free of any productsand/or infectious agents comprised in animal sera. In contrast,compositions comprising viruses produced according to conventionalmethods comprise residual compounds derived from animal serum. This isespecially the case for compositions comprising poxviruses producedaccording to conventional methods, such as vaccinia virus strains.

The invention further relates to the use of a virus as defined above ora composition as defined above for the manufacture of a vaccine.

The invention further concerns a method for the treatment or vaccinationof an animal, including a human, in need thereof, comprising theadministration of a virus as defined above or a composition as definedabove to the animal or human body.

EXPERIMENTAL PART

The present invention will be better understood in connection with thefollowing examples, which are intended as an illustration of and not alimitation upon the scope of the invention.

Example 1 Preparation of Chicken Embryo Fibroblast (CEF) Cells

Specific pathogen free (SPF) fertilized eggs were stored not longer than12 days at 4° C. The eggs were put into an incubator and incubated for10-12 days at 37.8° C.±8° C. One petri dish per maximum 11 eggs wasprepared with 10-20 ml PBS. The eggs were put in a dedicated egg cartonand treated extensively with Bacillol® to sterilize the outside of theegg shell. After drying, a hole was made into the eggs and the shell wasremoved carefully. The chorioallantoic membrane was put aside. Theembryos were lifted up by the feet and then their heads were cut off.The embryos were then transferred into the prepared petri dishes. Afterremoving the feet the trunks were washed again with PBS. 11 trunksmaximum were put into a 20 ml plastic syringe and squeezed into anErlenmeyer flask. 5 ml of prewarmed (37° C.) Trypsin/EDTA-solution pertrunk were added and the solution was stirred for 15 minutes with serumfree medium at room temperature using a magnetic stirrer. Trypsinizedcells were poured through a layer of mesh into a beaker. All cells weretransferred to one 225 ml-centrifuge tube and centrifuged down at 20°C., 470×g for 7 minutes in a bench top centrifuge. After discarding thesupernatant, the pellet was resuspended in 1 ml fresh pre-warmed (37°C.) serum free growth medium comprising 10 ng/ml EGF per trunk bypipetting up and down thoroughly. Fresh prewarmed (37° C.) serum freegrowth medium comprising 10 ng/ml EGF was added to a total volume of 150ml. The centrifugation step was repeated. The supernatant was removedand the pellet was resuspended as described above. Fresh prewarmed (37°C.) serum free growth medium comprising 10 ng/ml EGF was added to atotal volume of 100 ml. Cells were counted as described in the followingsection. The required amounts of cells were seeded in roller bottleswith serum free growth medium comprising 10 ng/ml EGF and incubated at37° C. Cells were ready for virus infection at day four after seeding.

Example 2 Counting Cell Density

A sample of the cell suspension (see section CEF preparation) was takenand mixed with one volume of Trypan blue, resulting in a final cellcount of 20 to 100 cells per 16 small squares of a hemocytometersupplied by Fuchs-Rosenthal under the name of Hemocytometer Fast Read102 (1:2-1:10 dilution). The sample was taken immediately afterresuspending the cells in order to prevent reaggregation orsedimentation of the cells. After a few minutes of incubation time withTrypan blue in order to get the dye properly into dead cells, 10 μl ofthe cell suspension was added to the hemocytometer. Only white, livingcells were counted under a light microscope using a 10× objective. Intotal, 3 representative big squares consisting of 3×16 small ones werecounted. From every big square only two borders in L-Form were includedin the counting. The average of counted cells was taken and the finalcell concentration was calculated using the following formula: Averagecell number×dilution×10⁴=cells/ml. Finally the cell suspension wasdiluted to the desired working concentration.

Example 3 Effect of the Addition of a Factor Selected from GrowthFactors and Fibronectin to a Serum Free Culture Medium on the Formationof a CEF-Monolayer

In preliminary experiments it was shown that CEF cells do not attach tothe surface of cell culture vessels if medium 199 is used that does notcomprise FCS. Moreover, no monolayers are formed. Normal monolayerformation is observed if medium 199 containing 7% FCS is used. It wasanalyzed whether attachment and growth of CEF cells in serum free medium199 can be achieved if recombinant Epidermal Growth Factor (r-hEGF) andFibronectin (FN) are added to the medium.

For the experiments CEF cells were grown in medium 199 with thedifferent additives alone or in combination. Cells grown in medium 199without any additives served as negative control. Cells cultivated inmedium 199 comprising 7% FCS served as positive control. All experimentswere conducted in 6-well cell culture plates with 3 ml medium. Theadditives were treated according to the data sheets of the supplierbefore being used for the cell culture. Fibronectin was allowed toadsorb to the surface of the cell culture plates for 25 minutes beforeuse. Fibronectin was used in a concentration of 3 μg/cm² and EGF wasused in a concentration of 10 ng/ml. Before adding any cells the cellculture plates were brought into contact with the fibronectin-containingmedium for 25 minutes.

Every culture medium plus the additives to be tested was cultured induplicate. The 6-well cell culture plates were incubated for 4 days at37° C. From day 1 to 4 the attachment and growth of the cells wasevaluated using a microscope.

For the positive control a normal attachment and growth of the CEF cellshas been observed. For Medium 199 without additives nearly no attachmentof CEF cells could be observed.

A crucial improvement in the forming of a monolayer was seen by the useof EGF added to Medium 199 compared to Medium 199 without additives. Itwas found that the cells attached and formed the typical fibroblastmorphology. Furthermore, a continuous growth could be observed over thewhole period of 4 days.

An improvement of cell attachment was also achieved by addingfibronectin to the culture medium. The addition of both, EGF andFibronectin resulted in a slight improvement compared to the addition ofEGF only and Fibronectin only.

In summary, monolayer formation of CEF cells in the serum-free Medium199 can be supported by the use of the additives EGF and Fibronectin.

Moreover, in parallel sets of experiments 1×10⁷ CEF cells were seeded inmedium comprising 10% FCS, medium not comprising FCS and medium notcomprising FCS but comprising EGF. The cell number was counted 2 daysafter seeding. The number of cells amounted to 42%, 6% and 44%,respectively, of the cell number used for seeding. Thus, the results forthe cells seeded in serum free medium comprising EGF were as good as theresults obtained with medium comprising FCS and significantly betterthan with medium neither containing serum nor EGF.

In addition the medium comprising EGF was compared to various standardserum free media, such as DMEM, Opti-Mem or 293-SFM. To this end 1×10⁷CEF cells were seeded in the various serum-free media and cultivated for4 days. The number of cells cultivated in medium comprising EGF was 24,5 and 12 times higher than the number of cells cultivated in serum freeDMEM, Opti.Mem and 293-SFM, respectively.

Example 4 Infection of CEF Cells with MVA

CEF cells were infected four days after seeding in roller bottles. Atthat time point cells have grown to an adequate monolayer. Cells wereinfected with a MOI of 1 or 0.1 MVA. For the infection the growth mediumwas removed from the flasks. The desired amount of virus per rollerbottle was diluted in 20 ml of the appropriate infection medium withoutserum. At this stage the serum free medium may or may not comprise afactor selected from growth factors and fibronectin. Cells wereincubated with the virus for 1 hour at 37° C. at 0.3-0.5 rpm in a rollerbottle incubator. After 1 hour the roller bottles were filled with theappropriate serum free growth medium to a total volume of 200 ml perroller bottle. At this stage the serum free medium may or may notcomprise a factor selected from growth factors and fibronectin. Virusreplication was stopped after 48 or 72 hours by freezing the rollerbottles to −20° C.

Example 5 Preparation of Viral Extracts from Infected CEF Cells andTitration of MVA

The frozen roller bottles were thawed at room temperature. During thethawing process the cells detach from the surface of the roller bottlesand can mechanically be removed by shaking the flasks. Virus/cellsuspension was harvested and aliquoted to smaller volumes. To releasethe virus from the infected cells, virus/cell suspensions were 3 timesfreeze/thawed. The freeze/thawed virus samples were used for titration.

Titrations were performed on 1^(st) passage CEF cells in 96-well plates,using 10-fold dilutions of viral suspension and 8 replicates perdilution. After the infection, infected cells were visualized with ananti-vaccinia virus antibody and an appropriate staining solution.

In detail, at day zero of the assay primary CEF cells (see section“preparation of Chicken Embryo Fibroblast (CEF) cells”) were trypsinizedand counted as described in the section “counting cell density”. Thecells were diluted to 1×10⁵ cells/ml in RPMI medium with 7% FCS.Following this dilution, 100 μl were seeded in each well of the 96-wellplates using a multichannel pipette. Cells were incubated over night at37° C. and 5% CO₂. The virus samples to be titrated (see section“preparation of viral extracts from infected CEF cells) were seriallydiluted in 10-fold steps from 10⁻¹-10⁻¹² using RPMI without serum. Thisserial dilution is carried out by adding 900 μl RPMI to all the wells ofa 96-deep-well plate. 100 μl of virus sample was added to all the wellsof the first row and mixed. Thereafter, 100 μl of each sample weretransferred to the next row of wells using a multi-channel pipette. The96-deep-well plates were kept on ice when performing the dilutions.Plates were incubated for 5 days at 37° C. and 5% CO₂ to allow theinfection to proceed. After 5 days, cells were immunohistochemicallystained with a vaccinia virus specific antibody. For the staining, theculture medium was removed by turning the 96-well plate upside down overa receptacle. Cells were fixed with 100 μl/well methanol/acetone (1:1)mixture for 10 minutes at room temperature. The fixing solution wasremoved and plates were air-dried. After drying, cells were washed oncewith PBS and incubated for 1 hour at room temperature with theanti-vaccinia virus antibody (Anti-Vaccinia virus antibody, rabbitpolyclonal, IgG fraction (Quartett, Berlin, Germany #9503-2057) dilutedto 1:1000 in PBS with 3% FCS. After removing the antibody, cells werewashed twice with PBS and incubated for 1 hour at room temperature withHRP-coupled (Horseradish Peroxidase-coupled) anti-rabbit antibody(Anti-rabbit IgG antibody, HRP-coupled goat polyclonal (Promega,Mannheim, Germany # W4011) diluted to 1:1000 in PBS with 3% FCS. Again,cells were washed with PBS and stained either with o-Dianisidine or TMB.For using the o-Dianisidine staining method, cells were incubated with100 μl/well staining solution consisting of 5 mg o-Dianisidine and 180μl 30% H₂O₂ per 60 ml of 50 mM phosphate-citrate buffer. Cells wereincubated at room temperature until they stained brown. Infected cellswere clearly visible after 1-3 hours. Using the TMB staining method,cells were incubated with 30 μl/well 1.2 mM TMB (Seramun DiagnosticaGmbH). After 15 minutes incubation time, the TMB solution was removedand cells were washed once with PBS. Infected cells appear dark blue.The plates were scored for infected cells. The viral titer wascalculated using the formula of Spearman and Kaerber. For thecalculation of the TCID₅₀ every well showing brown or blue cells wasmarked positive. Because assay parameters are kept constant, thefollowing simplified formula was used:

Virus titer[TCID₅₀/ml]=10^([a+1.5+xa/8+xb/8+xc/8])  ^(i.)

b. a=dilution factor of last column, in which all eight wells arepositive

c. x_(a)=number of positive wells in column a+1

d. x_(b)=number of positive wells in column a+2

e. x_(c)=number of positive wells in column a+3

Example 6 Optimal Seeding Density for CEF Cells in Serum Free Medium andOptimal Amount of MVA for Infection of CEF Cells

An optimal seeding cell density of 7.5×10⁷ cells/850 cm² (surface of oneroller flask) was determined for serum-free CEF growth. Cells were ableto build a good monolayer without forming big clumps at day four afterseeding and could be infected at this time point.

Experiments were carried out to determine the best level of viralinoculation and length of the infection for the maximum production ofMVA from CEF cells cultured in a serum-free process. CEF cells wereseeded at a density of 7.5×10⁷ cells/850 cm² in medium according to thepresent invention. At day 4 after seeding, cells were infected withdifferent amounts of MVA in the range of 0.05 to 1.0 TCID₅₀/cell of MVA.Best results were obtained with 0.1 TCID₅₀/cell of MVA.

Example 7 Optimal pH of Serum Free Medium for Culturing and Infectionwith MVA

MVA and other poxvirus infections are sensitive pH below 7.0. Poxvirusesare not stable at acid pH and it is recommended that purified poxvirusesare stored in a buffered solution above pH 7.0 to ensure stability andviral integrity upon storage as a liquid viral preparation. Experimentswere carried out to determine the effect on virus yield when carryingout infection at different starting pH. Roller bottles were seeded withCEF cells in the usually way in serum free medium comprising 10 ng/mlEGF plus 4 mM L-glutamine and cultured for 4 days. Cells were infectedwith MVA at 0.1 TCID₅₀/cell in serum free medium comprising 10 ng/ml EGFplus L-glutamine and 1 mM asparagine at different pH's ranging from 6.5to 9.0. At 72 hours post infection, the pH of the medium was measuredand viral yields were determined by titrating cell extracts in the usualmanner. The results are presented in the following table, which showsthe effect of initial pH of the medium at the start of the infection onvirus yield.

serum free medium comprising 10 ng/ml EGF Starting pH pH at 72 h p.i.Titer [TCID₅₀/ml] 6.5 7.05 0.56 × 10⁷ 7.0 7.34 10.0 × 10⁷ 7.5 7.53 5.60× 10⁷ 8.0 7.68 8.60 × 10⁷ 8.5 7.75 7.80 × 10⁷ 9.0 8.03 0.65 × 10⁷

For the infections carried out in serum free medium comprising 10 ng/mlEGF supplemented with L-glutamine and asparagine, the viral productionwas relatively constant with a starting pH from 7.0 to 8.5 but viralproductions were low at starting pH of 6.5 and 9.0. Best yield wasobtained at starting pH 7.0. Commercially available standard serum freemedia usually have a pH of 7.4. Therefore adjusting the pH of the serumfree medium to 7.0 can help to improve virus yield.

Example 8 Effect of Added Asparagine to the Serum Free Medium

Preliminary experiments have revealed that the amount of asparagine maybe limiting during the cultivation of CEF cells and the infection of CEFcells with MVA. To overcome the depletion of asparagine in the serumfree media during the culturing and infection process, extra asparaginewas added to the medium as a supplement before infecting CEF cells. Todetermine the optimal amount of asparagine to supplement the mediumwith, roller bottles were seeded with CEF cells (7.5×10⁷ cells/850 cm²)in serum free medium comprising 10 ng/ml EGF plus 4 mM L-glutamine. Fourdays after seeding cells were infected with MVA at 0.1 TCID₅₀/cell inserum free medium comprising 10 ng/ml EGF plus 4 mM L-glutaminesupplemented with different asparagine concentrations (0.5, 1.0 and 1.5mM). Viral replication was stopped at 72 hours post infection and viraltiters were determined. The results are shown in the following tablethat shows the production of MVA from CEF cells supplemented withdifferent levels of asparagine for the infection stage. The titersrepresent the averages of 3 roller bottles per asparaginesupplementation.

Supplement Viral titers after 72 hours infection Asparagine [TCID₅₀/ml]0.0 mM 1.8 × 10⁸ 0.5 mM 1.3 × 10⁸ 1.0 mM 6.8 × 10⁸ 1.5 mM 1.0 × 10⁸

The results demonstrate that supplementing the serum free mediumcomprising 10 ng/ml EGF medium with asparagine could improve viralproduction and that supplementation to 1 mM for the infection processwas optimal.

Example 9 Re-Derivation of Viruses

It is the aim of this example to show the usefulness of the methodsaccording to the present invention for the re-derivation of viruses. Wetherefore intentionally cultivate MVA-BN under standard serum containingconditions. Accordingly, such vaccine may potentially comprise undesiredviral contaminants or infectious agents such as BSE. The virus obtainedafter cultivation under serum containing conditions is then used asstarting material for the re-derivation of the virus under serum freeconditions according to methods described in the present application toobtain a re-derived virus stock wherein the risk of said virus tocontain a BSE particle is less than 10³².

MVA-BN virus seed stock: The starting material for a re-derived MVA-BNvirus seed stock is an inoculate obtained by intentionally cultivatingMVA-BN under standard serum containing conditions (10% fetal calfserum).

Primary CEF Cells: Primary CEF cells are prepared from certified SPFeggs as outlined below. Certified fertilised SPF eggs are supplied byCharles River SPAFAS. The flocks at Charles River are tested accordingto European Pharmacopoeia section 5.2.2 (REF 12.4). Upon arrival thepackage and the eggs are checked visually for damage and dirt. Damagedeggs are removed. The eggs are stored refrigerated for not longer than12 days at +2° C. to 8° C. Before incubation the eggs are disinfected byspraying with Mel Sept and put into an egg incubator. Incubation isperformed for 10 to 12 days (preferable 11 days) at 37.8° C.+/−0.8° C.and 60%+/−10% relative humidity.

Prior to cell preparation the eggs are transferred to a dedicated eggcarton and extensively treated with Mel Sept by spraying. The eggs areallowed to dry under a laminar flow.

The eggs are opened and the embryos are removed. Dead embryos andembryos showing deformations are excluded.

The heads and feet of the embryos are cut off.

Trunks are homogenised mechanically by squeezing them in a plasticsyringe.

Cells are incubated at room temperature with Trypsin/EDTA solution whilestirring.

Homogenised cells are poured through one layer of mesh and collected.

The homogenised cells are centrifuged. The supernatant is discarded andthe cell sediment is washed with a serum free medium according to thepresent invention.

The cells are pelleted again by centrifugation.

The supernatant is discarded and the cells are re-suspended in a serumfree medium according to the present invention.

Cells are counted and immediately seeded in a serum free medium inappropriate culture vessels.

Plaque purification and final amplification of selected clone: The 5rounds of plaque purification by limited dilution are conducted.

Seeding of Cells:

Primary CEF cells are seeded in a T175 flask (1×10⁷ cells/flask) in aserum free medium according to the present invention and incubated at37° C.+/−2° C. for 3 to 8 days.

First passage CEF cells are seeded in 96 well plates (1-2×10⁵ cells/ml)using a serum free medium according to the present invention andincubated for 24 h at 37° C.+/−2° C.

Approximately 10 plates are used per round of plaque purification.

Infection of Cells:

10 fold serial virus dilutions (10⁻¹ to 10⁻¹⁰) are prepared in a serumfree medium according to the present invention. 100 μl of the virusdilution/well are transferred to the 96 well plates containing CEFcells.

The plates are incubated for 5 to 6 days at 37° C.+/−2° C.

Isolation of Plaques:

Single virus plaques are visually detected under a microscope. 5 to 10single plaques are collected per round of plaque purification.

Each plaque is harvested using a pipette tip by scrapping andtransferred to a 1.5 ml tube. The volume is adjusted to 200 μl with aserum free medium according to the present invention.

The virus is released from the harvested cells by three cycles offreeze-thawing: freeze tube in liquid Nitrogen or at −80° C., thaw atroom temperature, repeat procedure twice.

The virus suspension can be stored at −80° C. until further analysis.Alternatively if only one single virus plaque is detected per well,cells can be harvested by freeze-thawing the whole 96 well platethree-times.

For amplification, 100 μl of the virus suspension is transferred tocells grown in 12 well plates.

Amplification of Virus on 12 Well Plates:

First passage CEF cells are seeded in 12 well plates (5×10⁴ cells/cm²)in 1 ml of a serum free medium according to the present invention andincubated for 24 h at 37° C.+/−2° C.

Cells show 80 to 100% confluence for infection.

100 μl of the virus suspension are added/well.

Cells are incubated for 48 to 72 h.

After 48 to 72 h the medium is removed and 300 μl of PBS per well areadded. Cells are harvested in PBS and transferred to a 1.5 ml tube. Ifcells are already detached they are harvested (by scraping) directly inthe media and transferred to a 1.5 ml tube.

The virus is released from the harvested cells by three cycles offreeze-thawing: freeze tube in liquid Nitrogen or at −80° C., thaw atroom temperature, repeat procedure twice.

The virus suspension can be stored at −80° C. until further analysis.

Screening of Amplified Virus:

200 μl of the solution are used for DNA preparation and PCR screening.

The remaining 100 μl are used for the next plaque purification round.

Final amplification of selected clone: After 5 rounds of plaquepurification, the final selected clone is further amplified to obtainenough material to produce a new master seed. The minimal amount ofvirus needed for production of a new master seed is 1×10⁸ TCID₅₀ in16-20 ml. The selected clone (already amplified on a 12 well plate) istransferred to a T25 cell culture flask for amplification. The cellvirus suspension is harvested by three cycles of freeze-thawing. Thevirus suspension is then transferred to a T75 cell culture flask foramplification and harvested. The virus is released by 3 cycles offreeze-thawing. Final amplification is performed in 3 to 5 T175 cellculture flasks. Material from 3 to 5 T175 flasks is harvested andsubjected to 3 cycles of freeze-thawing. The virus suspension istitrated, checked for sterility and tested for identity by PCR analysisof the 6 deletion sites.

Production of new master seed: Primary CEF cells are seeded in rollerbottles (850 cm²) with 7.5×10⁷ CEF cells in 200 ml of a serum freemedium according to the present invention. 2 to 5 roller bottles areseeded and incubated for 4 days at 37° C.+/−2° C., 0.3 rpm (±0.2 rpm) ina roller incubator. A virus suspension is prepared with a final titer of1.0×1×10⁶ TCID₅₀ (±0.5 log) in RPMI media. 10 ml are needed per rollerbottle. This corresponds to an MOI of 0.1. The medium is removed fromthe roller bottles. 10 ml of the virus suspension is added to eachroller bottle and incubated for 1-3 hrs in a roller incubator. 140 mlRPMI are added and incubated for 72 hours (±8 hours), 0.5 rpm (±0.2rpm).

Bottles are checked macroscopically for microbial contamination. Theroller bottles are transferred into a −20° C. freezer, and thecell/virus suspension is allowed to freeze. The roller bottles arestored at room temperature until the suspension has started to thaw andremove cells from the wall by shaking thoroughly. The cell/virussuspension is allowed to thaw completely. The cell/virus suspension isharvested into an appropriate vessel and aliquot a 4.5 ml in 5 mlcryotubes. Approximately 100 vials can be obtained from one rollerbottle. Filled virus suspension is stored at −20° C.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description.

All patents, applications, publications, test methods, literature, andother materials cited herein are hereby incorporated by reference.

1-47. (canceled)
 48. A method for the preparation of chicken embryofibroblast cells comprising: a) digesting a chicken embryo with aprotease to obtain chicken embryo fibroblast cells; and b) cultivatingthe chicken embryo fibroblast cells in a serum free medium comprisingepidermal growth factor at a concentration of 1-50 ng/ml.
 49. The methodof claim 48, wherein the serum free medium comprises a fibronectin. 50.The method of claim 48, wherein the epidermal growth factor is humanepidermal growth factor.
 51. The method of claim 50, wherein theepidermal growth factor is recombinant human epidermal growth factor ata concentration of 5-50 ng/ml.
 52. The method of claim 51, wherein theepidermal growth factor is recombinant human epidermal growth factor ata concentration of 5-20 ng/ml.
 53. The method of claim 52, wherein theserum free medium comprises human plasma fibronectin at a concentrationof 1-10 μg/cm².
 54. The method of claim 52, wherein the serum freemedium comprises a microbial extract, a plant extract, or an extractfrom a non-mammalian animal.
 55. The method of claim 52, wherein theserum free medium comprises asparagine in the range of 0.8-1.8 mM. 56.The method of claim 52, wherein the serum free medium comprisesglutamine in the range of 1-5 mM.
 57. The method of claim 48, whereinthe chicken embryo fibroblast cells are cultivated as adherent cellsattached to a solid support.
 58. The method of claim 52, wherein thechicken embryo fibroblast cells are cultivated as adherent cellsattached to a solid support.
 59. The method of claim 48, wherein thechicken embryo fibroblast cells are cultivated in suspension culture.60. The method of claim 52, wherein the chicken embryo fibroblast cellsare cultivated in suspension culture.
 61. The method of claim 48,wherein the CEFs are cultivated for 4 days in the serum free medium. 62.The method of claim 52, wherein the CEFs are cultivated for 4 days inthe serum free medium.
 63. The method of claim 57, wherein the CEFs arecultivated for 4 days in the serum free medium.
 64. The method of claim58, wherein the CEFs are cultivated for 4 days in the serum free medium.65. The method of claim 59, wherein the CEFs are cultivated for 4 daysin the serum free medium.
 66. The method of claim 60, wherein the CEFsare cultivated for 4 days in the serum free medium.
 67. The method ofclaim 48, wherein the protease is trypsin.